A 2-ketogluconate kinase KguK in Pseudomonas plecoglossicida JUIM01: Enzymatic characterization and its role in 2-keto-d-gluconic acid metabolism.

Abstract Our previous study has revealed that Pseudomonas plecoglossicida JUIM01 produced 2-keto- d -gluconic acid (2KGA) and re-utilized 2KGA as an alternative carbon source to support cell growth after complete consumption of glucose. Phosphorylation of intracellular 2KGA to 2-keto-6-phosphogluconic acid by 2-ketogluconate kinase (KguK) was regarded as the first step of 2KGA catabolism in Pseudomonas cytoplasm. In the present study, a kguK gene encoding 2-ketogluconate kinase from P. plecoglossicida JUIM01 was cloned and heterologously expressed in Pichia pastoris. The recombinant KguK showed the highest activity at 30–33 °C and pH 7.7, and high stability at 33 °C. Under the optimal conditions of 30 °C and pH 7.7 with addition of 5 mM Mg2+, the purified and concentrated (~30 folds) KguK had a specific activity of 3649.6 U/g and a Michaelis constant for 2KGA of 8.7 × 10−4 M. Knockout of kguK could retard but not completely inhibit 2KGA catabolism, indicating other existing 2KGA utilization pathway(s). The kguK-knockout P. plecoglossicida significantly reduced 2KGA re-utilization without negative effects on cell growth, glucose consumption or 2KGA production. The outputs demonstrated kguK knockout could be an effective strategy to develop the alternative 2KGA high-producing Pseudomonas strains to avoid the decrease of 2KGA yield caused by its re-utilization.